ATP synthase subunit homolog

ABSTRACT

The invention provides a human ATP synthase subunit homolog (ASYNT) and polynucleotides which identify and encode ASYNT. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating or preventing disorders associated with expression of ASYNT.

This application is a divisional application of U.S. application Ser.No. 09/154,802, filed Sep. 17, 1998.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of anATP synthase subunit homolog and to the use of these sequences in thediagnosis, treatment, and prevention of neurodegenerative disorders,myopathies, cancers, and immune disorders.

BACKGROUND OF THE INVENTION

Most cellular ATP is synthesized in the mitochondria through the processof oxidative phosphorylation. The mitochondrial electron transport orrespiratory chain is a series of enzyme complexes in the mitochondrialmembrane that is responsible for the transport of electrons from NADH tooxygen and the coupling of this oxidation to the synthesis of ATP(oxidative phosphorylation). ATP then provides the primary source ofenergy for driving a cell's many energy-requiring reactions. Theactivities of the enzymes of the mammalian oxidative phosphorylationsystem vary greatly in response to a number of physiological conditions,including cell proliferation, hormonal stimulation, development, anddifferentiation.

ATP synthase (F₁ F₀ ATPase) is the enzyme complex which serves as areversible coupling device that interconverts the energies of anelectrochemical proton gradient across the mitochondrial membrane intoeither the synthesis or hydrolysis of ATP. This gradient is produced byother enzymes of the respiratory chain in the course of electrontransport from NADH to oxygen. When the cell's energy demands are high,electron transport from NADH to oxygen generates an electrochemicalgradient across the mitochondrial membrane. Proton translocation fromthe outer to the inner side of the membrane drives the synthesis of ATP.Under conditions of low energy requirements, and when there is an excessof ATP, this electrochemical gradient is reversed and ATP synthasehydrolyzes ATP. The energy of ATP hydrolysis is used to pump protons outof the mitochondrial matrix.

ATP synthase is a complex composed of two structurally and functionallydistinct sectors termed F₁ and F₀ The F₁ sector is bound at the membranesurface and functions as the catalytic portion which synthesizes orhydrolyzes ATP. The F₀ sector is the transmembrane proton carrier orpump and spans the mitochondrial membrane. Subunits α, β, γ, δ, ε, andan ATPase inhibitor protein, IF₁, comprise the Dglobular catalytic F₁portion of the complex. Subunits a, b, c, d, e, f, g, F6, OSCP, and A6Lcomprise the proton-translocating, membrane spanning F₀ portion of thecomplex. A stalk portion of 40-50 angstroms in length connects the F₁and F₀ portions together and serves to transfer electrochemical energybetween them. Subunits from both portions of the ATP synthase complexcontribute to the stalk. They are theγ, δ, and ε subunits from F₁, andF6, OSCP, b, and d subunits from F₀. The mammalian ATP synthase complexcomprises at least 16 subunits encoded by nuclear DNA and 2 subunits(subunit A6L and α) encoded by mitochondrial DNA (Belogrudov, G. I., etal. (1996) J. Biol. Chem. 271:20340-20345.)

A mutation in the ATP synthase subunit A6L gene has been geneticallylinked to maternally inherited genetic diseases including neurologicalmuscle weakness and cases of subacute necrotizing encephalopathy. Toinvestigate the biochemical effects of an ATP synthase subunit A6Lleu156→arg mutation on F₁ F₀ -ATP synthase, a leu207→arg mutation wasconstructed in F₁ F₀ -ATP synthase from E. coli. Characterization of themutated enzyme revealed that the mutation abolished detectable ATPsynthesis via oxidative phosphorylation. The leu 207→arg mutationresults in a structural alteration that blocks proton translocationthrough F₁ F₀ -ATP synthase (Hartzog P. E. and Cain B. D. (1993) J.Biol. Chem. 268:12250-12252.)

The discovery of a new ATP synthase subunit homolog and thepolynucleotides encoding it satisfies a need in the art by providing newcompositions which are useful in the diagnosis, prevention, andtreatment of neurodegenerative disorders, myopathies, cancer, and immunedisorders.

SUMMARY OF THE INVENTION

The invention is based on the discovery of a new human ATP synthasesubunit homolog (ASYNT), the polynucleotides encoding ASYNT, and the useof these compositions for the diagnosis, treatment, or prevention ofneurodegenerative disorders, myopathies, cancers, and immune disorders.

The invention features a substantially purified polypeptide comprisingthe amino acid sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1.

The invention further provides a substantially purified variant havingat least 90% amino acid sequence identity to the amino acid sequence ofSEQ ID NO: 1 or a fragment of SEQ ID NO: 1. The invention also providesan isolated and purified polynucleotide encoding the polypeptidecomprising the amino acid sequence of SEQ ID NO: 1 or a fragment of SEQID NO: 1. The invention also includes an isolated and purifiedpolynucleotide variant having at least 70% polynucleotide sequenceidentity to the polynucleotide encoding the polypeptide comprising theamino acid sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1.

The invention further provides an isolated and purified polynucleotidewhich hybridizes under stringent conditions to the polynucleotideencoding the polypeptide comprising the amino acid sequence of SEQ IDNO: 1 or a fragment of SEQ ID NO: 1, as well as an isolated and purifiedpolynucleotide having a sequence which is complementary to thepolynucleotide encoding the polypeptide comprising the amino acidsequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1.

The invention also provides an isolated and purified polynucleotidecomprising the polynucleotide sequence of SEQ ID NO:2 or a fragment ofSEQ ID NO:2, and an isolated and purified polynucleotide variant havingat least 70% polynucleotide sequence identity to the polynucleotidecomprising the polynucleotide sequence of SEQ ID NO:2 or a fragment ofSEQ ID NO:2. The invention also provides an isolated and purifiedpolynucleotide having a sequence complementary to the polynucleotidecomprising the polynucleotide sequence of SEQ ID NO:2 or a fragment ofSEQ ID NO:2.

The invention also provides a method for detecting a polynucleotide in asample containing nucleic acids, the method comprising the steps of (a)hybridizing the complement of the polynucleotide sequence to at leastone of the polynucleotides of the sample, thereby forming ahybridization complex; and (b) detecting the hybridization complex,wherein the presence of the hybridization complex correlates with thepresence of a polynucleotide in the sample. In one aspect, the methodfurther comprises amplifying the polynucleotide prior to hybridization.

The invention further provides an expression vector containing at leasta fragment of the polynucleotide encoding the polypeptide comprising thesequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1. In anotheraspect, the expression vector is contained within a host cell.

The invention also provides a method for producing a polypeptide, themethod comprising the steps of: (a) culturing the host cell containingan expression vector containing at least a fragment of a polynucleotideunder conditions suitable for the expression of the polypeptide; and (b)recovering the polypeptide from the host cell culture.

The invention also provides a pharmaceutical composition comprising asubstantially purified polypeptide having the sequence of SEQ ID NO: 1or a fragment of SEQ ID NO: 1 in conjunction with a suitablepharmaceutical carrier.

The invention further includes a purified antibody which binds to apolypeptide comprising the sequence of SEQ ID NO: 1 or a fragment of SEQID NO: 1, as well as a purified agonist and a purified antagonist of thepolypeptide.

The invention also provides a method for treating or preventing adisorder associated with decreased expression or activity of ASYNT, themethod comprising administering to a subject in need of such treatmentan effective amount of a pharmaceutical composition comprising asubstantially purified polypeptide having the amino acid sequence of SEQID NO: 1 or a fragment of SEQ ID NO: 1, in conjunction with a suitablepharmaceutical carrier.

The invention also provides a method for treating or preventing adisorder associated with increased expression or activity of ASYNT, themethod comprising administering to a subject in need of such treatmentan effective amount of an antagonist of the polypeptide having the aminoacid sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1.

BRIEF DESCRIPTION OF THE FIGURES AND TABLE

FIGS. 1A, 1B, and 1C show the amino acid sequence (SEQ ID NO: 1) andnucleic acid sequence (SEQ ID NO:2) of ASYNT. The alignment was producedusing MACDNASIS PRO software (Hitachi Software Engineering, S. SanFrancisco Calif.).

FIGS. 2A and 2B show the amino acid sequence alignment between ASYNT(1887516; SEQ ID NO: 1) and Yarrowia lipolytica mitochondrial ATPsynthase 6 (GI 1006572; SEQ ID NO:3), produced using the multisequencealignment program of LASERGENE software (DNASTAR. Madison Wis.).

Table 1 shows the programs, their descriptions, references, andthreshold parameters used to analyze ASYNT.

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular machines, materials and methods described, as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms "a," "an," and "the" include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to "ahost cell" includes a plurality of such host cells, and a reference to"an antibody" is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any machines,materials, and methods similar or equivalent to those described hereincan be used to practice or test the present invention, the preferredmachines, materials and methods are now described. All publicationsmentioned herein are cited for the purpose of describing and disclosingthe cell lines, protocols, reagents and vectors which are reported inthe publications and which might be used in connection with theinvention. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

DEFINITIONS

"ASYNT" refers to the amino acid sequences of substantially purifiedASYNT obtained from any species, particularly a mammalian species,including bovine, ovine, porcine, murine, equine, and preferably thehuman species, from any source, whether natural, synthetic,semi-synthetic, or recombinant.

The term "agonist" refers to a molecule which, when bound to ASYNT,increases or prolongs the duration of the effect of ASYNT. Agonists mayinclude proteins, nucleic acids, carbohydrates, or any other moleculeswhich bind to and modulate the effect of ASYNT.

An "allelic variant" is an alternative form of the gene encoding ASYNT.Allelic variants may result from at least one mutation in the nucleicacid sequence and may result in altered mRNAs or in polypeptides whosestructure or function may or may not be altered. Any given natural orrecombinant gene may have none, one, or many allelic forms. Commonmutational changes which give rise to allelic variants are generallyascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

"Altered" nucleic acid sequences encoding ASYNT include those sequenceswith deletions, insertions, or substitutions of different nucleotides,resulting in a polypeptide the same as ASYNT or a polypeptide with atleast one functional characteristic of ASYNT. Included within thisdefinition are polymorphisms which may or may not be readily detectableusing a particular oligonucleotide probe of the polynucleotide encodingASYNT, and improper or unexpected hybridization to allelic variants,with a locus other than the normal chromosomal locus for thepolynucleotide sequence encoding ASYNT. The encoded protein may also be"altered," and may contain deletions, insertions, or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent ASYNT. Deliberate amino acid substitutions maybe made on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues, as long as the biological or immunological activity of ASYNTis retained. For example, negatively charged amino acids may includeaspartic acid and glutamic acid, positively charged amino acids mayinclude lysine and arginine, and amino acids with uncharged polar headgroups having similar hydrophilicity values may include leucine,isoleucine, and valine; glycine and alanine; asparagine and glutamine;serine and threonine; and phenylalanine and tyrosine.

The terms "amino acid" or "amino acid sequence" refer to anoligopeptide, peptide, polypeptide, or protein sequence, or a fragmentof any of these, and to naturally occurring or synthetic molecules. Inthis context, "fragments," "immunogenic fragments," or "antigenicfragments" refer to fragments of ASYNT which are preferably at least 5to about 15 amino acids in length, most preferably at least 14 aminoacids, and which retain some biological activity or immunologicalactivity of ASYNT. Where "amino acid sequence" is recited to refer to anamino acid sequence of a naturally occurring protein molecule, "aminoacid sequence" and like terms are not meant to limit the amino acidsequence to the complete native amino acid sequence associated with therecited protein molecule.

"Amplification" relates to the production of additional copies of anucleic acid sequence. Amplification is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art.

The term "antagonist" refers to a molecule which, when bound to ASYNT,decreases the amount or the duration of the effect of the biological orimmunological activity of ASYNT. Antagonists may include proteins,nucleic acids, carbohydrates, antibodies, or any other molecules whichdecrease the effect of ASYNT.

The term "antibody" refers to intact molecules as well as to fragmentsthereof, such as Fab, F(ab')₂, and Fv fragments, which are capable ofbinding the epitopic determinant. Antibodies that bind ASYNTpolypeptides can be prepared using intact polypeptides or usingfragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

The term "antigenic determinant" refers to that fragment of a molecule(i.e., an epitope) that makes contact with a particular antibody. When aprotein or a fragment of a protein is used to immunize a host animal,numerous regions of the protein may induce the production of antibodieswhich bind specifically to antigenic determinants (given regions orthree-dimensional structures on the protein). An antigenic determinantmay compete with the intact antigen (i.e., the immunogen used to elicitthe immune response) for binding to an antibody.

The term "antisense" refers to any composition containing a nucleic acidsequence which is complementary to the "sense" strand of a specificnucleic acid sequence. Antisense molecules may be produced by any methodincluding synthesis or transcription. Once introduced into a cell, thecomplementary nucleotides combine with natural sequences produced by thecell to form duplexes and to block either transcription or translation.The designation "negative" can refer to the antisense strand, and thedesignation "positive" can refer to the sense strand.

The term "biologically active," refers to a protein having structural,regulatory, or biochemical functions of a naturally occurring molecule.Likewise, "immunologically active" refers to the capability of thenatural, recombinant, or synthetic ASYNT, or of any oligopeptidethereof, to induce a specific immune response in appropriate animals orcells and to bind with specific antibodies.

The terms "complementary" or "complementarity" refer to the naturalbinding of polynucleotides by base pairing. For example, the sequence"5' A-G-T 3' " bonds to the complementary sequence "3' T-C-A 5'."Complementarity between two single-stranded molecules may be "partial,"such that only some of the nucleic acids bind, or it may be "complete,"such that total complementarity exists between the single strandedmolecules. The degree of complementarity between nucleic acid strandshas significant effects on the efficiency and strength of thehybridization between the nucleic acid strands. This is of particularimportance in amplification reactions, which depend upon binding betweennucleic acids strands, and in the design and use of peptide nucleic acid(PNA) molecules.

A "composition comprising a given polynucleotide sequence" or a"composition comprising a given amino acid sequence" refer broadly toany composition containing the given polynucleotide or amino acidsequence. The composition may comprise a dry formulation or an aqueoussolution. Compositions comprising polynucleotide sequences encodingASYNT or fragments of ASYNT may be employed as hybridization probes. Theprobes may be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., sodium dodecyl sulfate; SDS), and other components(e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).

"Consensus sequence" refers to a nucleic acid sequence which has beenresequenced to resolve uncalled bases, extended using XL-PCR kit(Perkin-Elmer, Norwalk Conn.) in the 5' and/or the 3' direction, andresequenced, or which has been assembled from the overlapping sequencesof more than one Incyte Clone using a computer program for fragmentassembly, such as the GELVIEW fragment assembly system (GCG, MadisonWis.). Some sequences have been both extended and assembled to producethe consensus sequence.

The term "correlates with expression of a polynucleotide" indicates thatthe detection of the presence of nucleic acids, the same or related to anucleic acid sequence encoding ASYNT, by northern analysis is indicativeof the presence of nucleic acids encoding ASYNT in a sample, and therebycorrelates with expression of the transcript from the polynucleotideencoding ASYNT.

A "deletion" refers to a change in the amino acid or nucleotide sequencethat results in the absence of one or more amino acid residues ornucleotides.

The term "derivative" refers to the chemical modification of apolypeptide sequence, or a polynucleotide sequence. Chemicalmodifications of a polynucleotide sequence can include, for example,replacement of hydrogen by an alkyl, acyl, or amino group. A derivativepolynucleotide encodes a polypeptide which retains at least onebiological or immunological function of the natural molecule. Aderivative polypeptide is one modified by glycosylation, pegylation, orany similar process that retains at least one biological orimmunological function of the polypeptide from which it was derived.

The term "similarity" refers to a degree of complementarity. There maybe partial similarity or complete similarity. The word "identity" maysubstitute for the word "similarity." A partially complementary sequencethat at least partially inhibits an identical sequence from hybridizingto a target nucleic acid is referred to as "substantially similar." Theinhibition of hybridization of the completely complementary sequence tothe target sequence may be examined using a hybridization assay(Southern or northern blot, solution hybridization, and the like) underconditions of reduced stringency. A substantially similar sequence orhybridization probe will compete for and inhibit the binding of acompletely similar (identical) sequence to the target sequence underconditions of reduced stringency. This is not to say that conditions ofreduced stringency are such that non-specific binding is permitted, asreduced stringency conditions require that the binding of two sequencesto one another be a specific (i.e., a selective) interaction. Theabsence of non-specific binding may be tested by the use of a secondtarget sequence which lacks even a partial degree of complementarity(e.g., less than about 30% similarity or identity). In the absence ofnon-specific binding, the substantially similar sequence or probe willnot hybridize to the second non-complementary target sequence.

The phrases "percent identity" or "% identity" refer to the percentageof sequence similarity found in a comparison of two or more amino acidor nucleic acid sequences. Percent identity can be determinedelectronically, e.g., by using the MEGALIGN program (DNASTAR) whichcreates alignments between two or more sequences according to methodsselected by the user, e.g., the clustal method. (See, e.g., Higgins, D.G. and P. M. Sharp (1988) Gene 73:237-244.) The clustal algorithm groupssequences into clusters by examining the distances between all pairs.The clusters are aligned pairwise and then in groups. The percentagesimilarity between two amino acid sequences, e.g. sequence A andsequence B, is calculated by dividing the length of sequence A, minusthe number of gap residues in sequence A, minus the number of gapresidues in sequence B, into the sum of the residue matches betweensequence A and sequence B, times one hundred. Gaps of low or of nosimilarity between the two amino acid sequences are not included indetermining percentage similarity. Percent identity between nucleic acidsequences can also be counted or calculated by other methods known inthe art, e.g., the Jotun Hein method. (See, e.g., Hein, J. (1990)Methods Enzymol. 183:626-645.) Identity between sequences can also bedetermined by other methods known in the art, e.g., by varyinghybridization conditions.

"Human artificial chromosomes" (HACs) are linear microchromosomes whichmay contain DNA sequences of about 6 kb to 10 Mb in size, and whichcontain all of the elements required for stable mitotic chromosomesegregation and maintenance.

The term "humanized antibody" refers to antibody molecules in which theamino acid sequence in the non-antigen binding regions has been alteredso that the antibody more closely resembles a human antibody, and stillretains its original binding ability.

"Hybridization" refers to any process by which a strand of nucleic acidbinds with a complementary strand through base pairing.

The term "hybridization complex" refers to a complex formed between twonucleic acid sequences by virtue of the formation of hydrogen bondsbetween complementary bases. A hybridization complex may be formed insolution (e.g., C₀ t or R₀ t analysis) or formed between one nucleicacid sequence present in solution and another nucleic acid sequenceimmobilized on a solid support (e.g., paper, membranes, filters, chips,pins or glass slides, or any other appropriate substrate to which cellsor their nucleic acids have been fixed).

The words "insertion" or "addition" refer to changes in an amino acid ornucleotide sequence resulting in the addition of one or more amino acidresidues or nucleotides, respectively, to the sequence found in thenaturally occurring molecule.

"Immune response" can refer to conditions associated with inflammation,trauma, immune disorders, or infectious or genetic disease, etc. Theseconditions can be characterized by expression of various factors, e.g.,cytokines, chemokines, and other signaling molecules, which may affectcellular and systemic defense systems.

The term "microarray" refers to an arrangement of distinctpolynucleotides on a substrate.

The terms "element" or "array element" in a microarray context, refer tohybridizable polynucleotides arranged on the surface of a substrate.

The term "modulate" refers to a change in the activity of ASYNT. Forexample, modulation may cause an increase or a decrease in proteinactivity, binding characteristics, or any other biological, functional,or immunological properties of ASYNT.

The phrases "nucleic acid" or "nucleic acid sequence" refer to anucleotide, oligonucleotide, polynucleotide, or any fragment thereof.These phrases also refer to DNA or RNA of genomic or synthetic originwhich may be single-stranded or double-stranded and may represent thesense or the antisense strand, to peptide nucleic acid (PNA), or to anyDNA-like or RNA-like material. In this context, "fragments" refers tothose nucleic acid sequences which, when translated, would producepolypeptides retaining some functional characteristic, e.g.,antigenicity, or structural domain characteristic, e.g., ATP-bindingsite, of the full-length polypeptide.

The terms "operably associated" or "operably linked" refer tofunctionally related nucleic acid sequences. A promoter is operablyassociated or operably linked with a coding sequence if the promotercontrols the translation of the encoded polypeptide. While operablyassociated or operably linked nucleic acid sequences can be contiguousand in the same reading frame, certain genetic elements, e.g., repressorgenes, are not contiguously linked to the sequence encoding thepolypeptide but still bind to operator sequences that control expressionof the polypeptide.

The term "oligonucleotide" refers to a nucleic acid sequence of at leastabout 6 nucleotides to 60 nucleotides, preferably about 15 to 30nucleotides, and most preferably about 20 to 25 nucleotides, which canbe used in PCR amplification or in a hybridization assay or microarray."Oligonucleotide" is substantially equivalent to the terms "amplimer,""primer," "oligomer," and "probe," as these terms are commonly definedin the art.

"Peptide nucleic acid" (PNA) refers to an antisense molecule oranti-gene agent which comprises an oligonucleotide of at least about 5nucleotides in length linked to a peptide backbone of amino acidresidues ending in lysine. The terminal lysine confers solubility to thecomposition. PNAs preferentially bind complementary single stranded DNAor RNA and stop transcript elongation, and may be pegylated to extendtheir lifespan in the cell.

The term "sample" is used in its broadest sense. A sample suspected ofcontaining nucleic acids encoding ASYNT, or fragments thereof, or ASYNTitself, may comprise a bodily fluid; an extract from a cell, chromosome,organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA,or cDNA, in solution or bound to a substrate; a tissue; a tissue print;etc.

The terms "specific binding" or "specifically binding" refer to thatinteraction between a protein or peptide and an agonist, an antibody, oran antagonist. The interaction is dependent upon the presence of aparticular structure of the protein, e.g., the antigenic determinant orepitope, recognized by the binding molecule. For example, if an antibodyis specific for epitope "A," the presence of a polypeptide containingthe epitope A, or the presence of free unlabeled A, in a reactioncontaining free labeled A and the antibody will reduce the amount oflabeled A that binds to the antibody.

The term "stringent conditions" refers to conditions which permithybridization between polynucleotides and the claimed polynucleotides.Stringent conditions can be defined by salt concentration, theconcentration of organic solvent. e.g., formamide, temperature. andother conditions well known in the art. In particular, stringency can beincreased by reducing the concentration of salt. increasing theconcentration of formamide, or raising the hybridization temperature.

The term "substantially purified" refers to nucleic acid or amino acidsequences that are removed from their natural environment and areisolated or separated, and are at least about 60% free, preferably about75% free, and most preferably about 90% free from other components withwhich they are naturally associated.

A "substitution" refers to the replacement of one or more amino acids ornucleotides by different amino acids or nucleotides, respectively.

"Substrate" refers to any suitable rigid or semi-rigid support includingmembranes, filters, chips, slides, wafers, fibers, magnetic ornonmagnetic beads, gels, tubing, plates, polymers, microparticles andcapillaries. The substrate can have a variety of surface forms, such aswells, trenches, pins, channels and pores, to which polynucleotides orpolypeptides are bound.

"Transformation" describes a process by which exogenous DNA enters andchanges a recipient cell. Transformation may occur under natural orartificial conditions according to various methods well known in theart, and may rely on any known method for the insertion of foreignnucleic acid sequences into a prokaryotic or eukaryotic host cell. Themethod for transformation is selected based on the type of host cellbeing transformed and may include, but is not limited to, viralinfection, electroporation, heat shock, lipofection, and particlebombardment. The term "transformed" cells includes stably transformedcells in which the inserted DNA is capable of replication either as anautonomously replicating plasmid or as part of the host chromosome, aswell as transiently transformed cells which express the inserted DNA orRNA for limited periods of time.

A "variant" of ASYNT polypeptides refers to an amino acid sequence thatis altered by one or more amino acid residues. The variant may have"conservative" changes, wherein a substituted amino acid has similarstructural or chemical properties (e.g., replacement of leucine withisoleucine). More rarely, a variant may have "nonconservative" changes(e.g., replacement of glycine with tryptophan). Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, LASERGENE software (DNASTAR).

The term "variant," when used in the context of a polynucleotidesequence, may encompass a polynucleotide sequence related to ASYNT. Thisdefinition may also include, for example, "allelic" (as defined above),"splice," "species," or "polymorphic" variants. A splice variant mayhave significant identity to a reference molecule, but will generallyhave a greater or lesser number of polynucleotides due to alternatesplicing of exons during mRNA processing. The corresponding polypeptidemay possess additional ftunctional domains or an absence of domains.Species variants are polynucleotide sequences that vary from one speciesto another. The resulting polypeptides generally will have significantamino acid identity relative to each other. A polymorphic variant is avariation in the polynucleotide sequence of a particular gene betweenindividuals of a given species. Polymorphic variants also may encompass"single nucleotide polymorphisms" (SNPs) in which the polynucleotidesequence varies by one base. The presence of SNPs may be indicative of,for example, a certain population, a disease state, or a propensity fora disease state.

THE INVENTION

The invention is based on the discovery of a new human ATP synthasesubunit homolog (ASYNT), the polynucleotides encoding ASYNT, and the useof these compositions for the diagnosis, treatment, or prevention ofneurodegenerative disorders, myopathies, cancer, and immune disorders.

Nucleic acids encoding the ASYNT of the present invention wereidentified in Incyte Clone 1887516 from the bladder tumor cDNA library(BLADTUT07) using a computer search for nucleotide and/or amino acidsequence alignments. A consensus sequence, SEQ ID NO:2, was derived fromthe following overlapping and/or extended nucleic acid sequences: IncyteClones 1887516F6 (BLADTUT07), 2325124H1 (OVARNOT02), 2545983F6(UTRSNOT11), and 2902529H1 (DRGCNOT01).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO: 1, as shown in FIG. 1A, 1B, and1C. ASYNT is 249 amino acids in length and has four potential caseinkinase phosphorylation sites at residues S90, S116, S146, and S232, andthree potential protein kinase C phosphorylation sites at residues S103,T182, and S218. HMM analysis indicates that ASTNT contains a potentialtransmembrane domain from residues M32 to F50, and SPSCAN indicates apotential hydrophobic domain from M1 to P58 As shown in FIGS. 2A and 2B,ASYNT has chemical and structural similarity with Yarrowia lipolyticamitochondrial ATP synthase 6 subunit (GI 1006572; SEQ ID NO:3). Inparticular, ASYNT and Yarrowia lipolytica mitochondrial ATP synthase 6subunit share 23% identity. A fragment of SEQ ID NO:2 from aboutnucleotide 119 to about nucleotide 160 is useful in hybridization oramplification technologies to identify SEQ ID NO:2 and to distinguishbetween SEQ ID NO:2 and a related sequence. The polypeptide encoded by afragment of SEQ ID NO:2 from about nucleotide 119 to about nucleotide160 is useful in Northern analysis shows the expression of this sequencein various libraries, at least 55% of which are associated with cellproliferative disorders. Of particular note is that 67% of the librariesin which ASYNT is expressed are derived from neural tissues.

The invention also encompasses ASYNT variants. A preferred ASYNT variantis one which has at least about 80%, more preferably at least about 90%,and most preferably at least about 95% amino acid sequence identity tothe ASYNT amino acid sequence, and which contains at least onefunctional or structural characteristic of ASYNT.

The invention also encompasses polynucleotides which encode ASYNT. In aparticular embodiment, the invention encompasses a polynucleotidesequence comprising the sequence of SEQ ID NO:2, which encodes an ASYNT.

The invention also encompasses a variant of a polynucleotide sequenceencoding ASYNT. In particular, such a variant polynucleotide sequencewill have at least about 70%, more preferably at least about 85%, andmost preferably at least about 95% polynucleotide sequence identity tothe polynucleotide sequence encoding ASYNT. A particular aspect of theinvention encompasses a variant of SEQ ID NO:2 which has at least about70%, more preferably at least about 85%, and most preferably at leastabout 95% polynucleotide sequence identity to SEQ ID NO:2. Any one ofthe polynucleotide variants described above can encode an amino acidsequence which contains at least one functional or structuralcharacteristic of ASYNT.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of polynucleotidesequences encoding ASYNT, some bearing minimal similarity to thepolynucleotide sequences of any known and naturally occurring gene, maybe produced. Thus, the invention contemplates each and every possiblevariation of polynucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe polynucleotide sequence of naturally occurring ASYNT, and all suchvariations are to be considered as being specifically disclosed.

Although nucleotide sequences which encode ASYNT and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring ASYNT under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding ASYNT or its derivatives possessing a substantially differentcodon usage, e.g., inclusion of non-naturally occurring codons. Codonsmay be selected to increase the rate at which expression of the peptideoccurs in a particular prokaryotic or eukaryotic host in accordance withthe frequency with which particular codons are utilized by the host.Other reasons for substantially altering the nucleotide sequenceencoding ASYNT and its derivatives without altering the encoded aminoacid sequences include the production of RNA transcripts having moredesirable properties, such as a greater half-life, than transcriptsproduced from the naturally occurring sequence.

The invention also encompasses production of DNA sequences which encodeASYNT and ASYNT derivatives, or fragments thereof, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents well known in the art. Moreover, synthetic chemistry may beused to introduce mutations into a sequence encoding ASYNT or anyfragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed polynucleotide sequences, and, inparticular, to those shown in SEQ ID NO:2, or to a fragment of SEQ IDNO:2, under various conditions of stringency. (See, e.g., Wahl, G. M.and S. L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A. R.(1987) Methods Enzymol. 152:507-511.) For example, stringent saltconcentration will ordinarily be less than about 750 mM NaCl and 75 mMtrisodium citrate, preferably less than about 500 mM NaCl and 50 mMtrisodium citrate, and most preferably less than about 250 mM NaCl and25 mM trisodium citrate. Low stringency hybridization can be obtained inthe absence of organic solvent, e.g., formamide, while high stringencyhybridization can be obtained in the presence of at least about 35%formamide, and most preferably at least about 50% formamide. Stringenttemperature conditions will ordinarily include temperatures of at leastabout 30° C., more preferably of at least about 37° C., and mostpreferably of at least about 42° C. Varying additional parameters, suchas hybridization time, the concentration of detergent, e.g., sodiumdodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA,are well known to those skilled in the art. Various levels of stringencyare accomplished by combining these various conditions as needed. In apreferred embodiment, hybridization will occur at 30° C. in 750 mM NaCl,75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment,hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodiumcitrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA(ssDNA). In a most preferred embodiment, hybridization will occur at 42°C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50 % formamide, and200 μg/ml ssDNA. Useful variations on these conditions will be readilyapparent to those skilled in the art.

The washing steps which follow hybridization can also vary instringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude temperature of at least about 25° C., more preferably of atleast about 42° C., and most preferably of at least about 68° C. In apreferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a most preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art.

Methods for DNA sequencing are well known in the art and may be used topractice any of the embodiments of the invention. The methods may employsuch enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE DNApolymerase (US Biochemical, Cleveland, OHiO.), Taq polymerase(Perkin-Elmer), thermostable T7 polymerase (Amersham Pharmacia Biotech,Piscataway, N.J.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE amplification system(Life Technologies, Gaithersburg, Md.). Preferably, sequence preparationis automated with machines such as the Robbins Hydra microdispenser(Robbins Scientific, Sunnyvale, Calif.), MICROLAB 2200 liquid transtersystem (Hamilton, Reno, Nev.), Peltier thermal cycler 200 (PTC200; MJResearch, Watertown, Mass.) and the ABI CATALYST 800 (Perkin-Elmer).Sequencing is then carried out using either ABI 373 or 377 DNAsequencing systems (Perkin-Elmer) or the MEGABACE 1000 DNA sequencingsystem (Molecular Dynamics, Sunnyvale, Calif.). The resulting sequencesare analyzed using a variety of algorithms which are well known in theart. (See, e.g., Ausubel, F. M. (1997) Short Protocols in MolecularBiology, John Wiley & Sons, New York, N.Y., unit 7.7; Meyers, R. A.(1995) Molecular Biology and Biotechnology, Wiley VCH, New York, N.Y.,pp. 856-853.)

The nucleic acid sequences encoding ASYNT may be extended utilizing apartial nucleotide sequence and employing various PCR-based methodsknown in the art to detect upstream sequences, such as promoters andregulatory elements. For example, one method which may be employed,restriction-site PCR, uses universal and nested primers to amplifyunknown sequence from genomic DNA within a cloning vector. (See, e.g.,Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) Another method,inverse PCR, uses primers that extend in divergent directions to amplifyunknown sequence from a circularized template. The template is derivedfrom restriction fragments comprising a known genomic locus andsurrounding sequences. (See, e.g., Triglia, T. et al. (1988) NucleicAcids Res. 16:8186.) A third method, capture PCR, involves PCRamplification of DNA fragments adjacent to known sequences in human andyeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al.(1991) PCR Methods Applic. 1:111-119.) In this method, multiplerestriction enzyme digestions and ligations may be used to insert anengineered double-stranded sequence into a region of unknown sequencebefore performing PCR. Other methods which may be used to retrieveunknown sequences are known in the art. (See, e.g., Parker, J. D. et al.(199 1) Nucleic Acids Res. 19:3055-306). Additionally, one may use PCR,nested primers, and PROMOTERINDER libraries (Clontech, Palo Alto,Calif.) to walk genomic DNA. This procedure avoids the need to screenlibraries and is useful in finding intron/exon junctions. For allPCR-based methods, primers may be designed using commercially availablesoftware, such as OLIGO 4.06 primer analysis software (NationalBiosciences, Plymouth, Minn.) or another appropriate program, to beabout 22 to 30 nucleotides in length, to have a GC content of about 50%or more, and to anneal to the template at temperatures of about 68° C.to 72° C.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. In addition,random-primed libraries, which often include sequences containing the 5'regions of genes, are preferable for situations in which an oligo d(T)library does not yield a full-length cDNA. Genomic libraries may beuseful for extension of sequence into 5' non-transcribed regulatoryregions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentnucleotide-specific, laser-stimulated fluorescent dyes, and a chargecoupled device camera for detection of the emitted wavelengths.Output/light intensity may be converted to electrical signal usingappropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR,Perkin-Elmer), and the entire process from loading of samples tocomputer analysis and electronic data display may be computercontrolled. Capillary electrophoresis is especially preferable forsequencing small DNA fragments which may be present in limited amountsin a particular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode ASYNT may be cloned in recombinant DNAmolecules that direct expression of ASYNT, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and used to express ASYNT.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alterASYNT-encoding sequences for a variety of purposes including, but notlimited to, modification of the cloning, processing, and/or expressionof the gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example,oligonucleotide-mediated site-directed mutagenesis may be used tointroduce mutations that create new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, and so forth.

In another embodiment, sequences encoding ASYNT may be synthesized, inwhole or in part, using chemical methods well known in the art. (See,e.g., Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.215-223, and Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser.225-232.) Alternatively, ASYNT itself or a fragment thereof may besynthesized using chemical methods. For example, peptide synthesis canbe performed using various solid-phase techniques. (See, e.g., Roberge,J. Y. et al. (1995) Science 269:202-204.) Automated synthesis may beachieved using the ABI 431A peptide synthesizer (Perkin-Elmer).Additionally, the amino acid sequence of ASYNT, or any part thereof, maybe altered during direct synthesis and/or combined with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

The peptide may be substantially purified by preparative highperformance liquid chromatography. (See, e.g, Chiez, R. M. and F. Z.Regnier (1990) Methods Enzymol. 182:392-421.) The composition of thesynthetic peptides may be confirmed by amino acid analysis or bysequencing. (See, e.g., Creighton, T. (1984) Proteins, Structures andMolecular Properties, W H Freeman, New York, N.Y.)

In order to express a biologically active ASYNT, the nucleotidesequences encoding ASYNT or derivatives thereof may be inserted into anappropriate expression vector, i.e., a vector which contains thenecessary elements for transcriptional and translational control of theinserted coding sequence in a suitable host. These elements includeregulatory sequences, such as enhancers, constitutive and induciblepromoters, and 5' and 3' untranslated regions in the vector and inpolynucleotide sequences encoding ASYNT. Such elements may vary in theirstrength and specificity. Specific initiation signals may also be usedto achieve more efficient translation of sequences encoding ASYNT. Suchsignals include the ATG initiation codon and adjacent sequences, e.g.the Kozak sequence. In cases where sequences encoding ASYNT and itsinitiation codon and upstream regulatory sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including an in-frame ATG initiation codonshould be provided by the vector. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers appropriate for the particular host cell system used. (See,e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding ASYNT andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, e.g., Sambrook, J.et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview, N.Y., ch. 4, 8, and 16-17; Ausubel, F. M. et al.(1995) Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, N.Y., ch. 9, 13, and 16.)

A variety of expression vector/host systems may be utilized to containand express sequences encoding ASYNT. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith viral expression vectors (e.g., baculovirus); plant cell systemstransformed with viral expression vectors (e.g., cauliflower mosaicvirus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

In bacterial systems, a number of cloning and expression vectors may beselected depending upon the use intended for polynucleotide sequencesencoding ASYNT. For example, routine cloning, subcloning, andpropagation of polynucleotide sequences encoding ASYNT can be achievedusing a multifunctional E coli vector such as PBLUESCRIPT (Stratagene,La Jolla, Calif.) or pSPORT1 plasmid (Life Technologies). Ligation ofsequences encoding ASYNT into the vector's multiple cloning sitedisrupts the lacZ gene, allowing a colorimetric screening procedure foridentification of transformed bacteria containing recombinant molecules.In addition, these vectors may be useful for in vitro transcription,dideoxy sequencing, single strand rescue with helper phage, and creationof nested deletions in the cloned sequence. (See, e.g., Van Heeke, G.and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) When largequantities of ASYNT are needed, e.g. for the production of antibodies,vectors which direct high level expression of ASYNT may be used. Forexample, vectors containing the strong, inducible T5 or T7 bacteriophagepromoter may be used.

Yeast expression systems may be used for production of ASYNT. A numberof vectors containing constitutive or inducible promoters, such as alphafactor, alcohol oxidase, and PGH promoters, may be used in the yeastSaccharomyces cerevisiae or Pichia pastoris. In addition, such vectorsdirect either the secretion or intracellular retention of expressedproteins and enable integration of foreign sequences into the hostgenome for stable propagation. (See, e.g., Ausubel, 1995, supra; Grantet al. (1987) Methods Enzymol. 153:516-54; and Scorer, C. A. et al.(1994) Bio/Technology 12:181-184.)

Plant systems may also be used for expression of ASYNT. Transcription ofsequences encoding ASYNT may be driven by viral promoters, e.g., the 35Sand 19S promoters of CaMV used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984)EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; andWinter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. (See, e.g., The McGrawHill Yearbook of Science and Technology (1992) McGraw Hill, New York,N.Y., pp. 191-196.)

In mammalian cells, a number of viral-based expression systems may beutilized. In cases where an adenovirus is used as an expression vector,sequences encoding ASYNT may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain infective virus whichexpresses ASYNT in host cells. (See, e.g., Logan, J. and T. Shenk (1984)Proc. Natl. Acad. Sci. 81:3655-3659.) In addition, transcriptionenhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used toincrease expression in mammalian host cells. SV40 or EBV-based vectorsmay also be used for high-level protein expression.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained in and expressed from aplasmid. HACs of about 6 kb to 10 Mb are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes. (See, e.g., Harrington, J. J. etal. (1997) Nat Genet. 15:345-355.)

For long term production of recombinant proteins in mammalian systems,stable expression of ASYNT in cell lines is preferred. For example,sequences encoding ASYNT can be transformed into cell lines usingexpression vectors which may contain viral origins of replication and/orendogenous expression elements and a selectable marker gene on the sameor on a separate vector. Following the introduction of the vector, cellsmay be allowed to grow for about 1 to 2 days in enriched media beforebeing switched to selective media. The purpose of the selectable markeris to confer resistance to a selective agent, and its presence allowsgrowth and recovery of cells which successfully express the introducedsequences. Resistant clones of stably transformed cells may bepropagated using tissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase and adenine phosphoribosyltransferase genes, for use intk or apr cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell11:223-232; Lowy, l. et al. (1980) Cell 22:817-823) Also,antimetabolite, antibiotic, or herbicide resistance can be used as thebasis for selection. For example, dhfr confers resistance tomethotrexate; neo confers resistance to the aminoglycosides, neomycinand G-4 18; and als and pat confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M.et al. (1980) Proc. Natl. Acad. Sci. 5 77:3567-3570; Colbere-Garapin, F.etal. (1981)J. Mol. Biol. 150:1-14.) Additional selectable genes havebeen described, e.g., trpB and hisD, which alter cellular requirementsfor metabolites. (See, e.g., Hartman, S. C. and R. C. Mulligan (1988)Proc. Natl. Acad. Sci. 85:8047-8051.) Visible markers, e.g.,anthocyanins, green fluorescent proteins (GFP; Clontech), βglucuronidaseand its substrate β-glucuronide, or luciferase and its substrateluciferin may be used. These markers can be used not only to to identifytransformants, but also to quantify the amount of transient or stableprotein expression attributable to a specific vector system. (See, e.g.,Rhodes, C. A. (1995) Methods Mol. Biol. 55:121-131.)

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, the presence and expression of thegene may need to be confirmed. For example, if the sequence encodingASYNT is inserted within a marker gene sequence, transformed cellscontaining sequences encoding ASYNT can be identified by the absence ofmarker gene function. Alternatively, a marker gene can be placed intandem with a sequence encoding ASYNT under the control of a singlepromoter. Expression of the marker gene in response to induction orselection usually indicates expression of the tandem gene as well.

In general, host cells that contain the nucleic acid sequence encodingASYNT and that express ASYNT may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCRamplification, and protein bioassay or immunoassay techniques whichinclude membrane, solution, or chip based technologies for the detectionand/or quantification of nucleic acid or protein sequences.

Immunological methods for detecting and measuring the expression ofASYNT using either specific polyclonal or monoclonal antibodies areknown in the art. Examples of such techniques include enzyme-linkedimmunosorbent assays (ELISAs), radioimmunoassays (RIAs), andfluorescence activated cell sorting (FACS). A two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes on ASYNT is preferred, but a competitivebinding assay may be employed. These and other assays are well known inthe art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, aLaboratory Manual, APS Press, St Paul, Minn., Sect. IV; Coligan, J. E.et al. (1997) Current Protocols in Immunology, Greene Pub. Associatesand Wiley-Interscience, New York, N.Y.; and Pound, J. D. (1998)Immunochemical Protocols, Humana Press, Totowa, N.J.).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding ASYNT includeoligolabeling, nick translation, end-labeling, or PCR amplificationusing a labeled nucleotide. Alternatively, the sequences encoding ASYNT,or any fragments thereof, may be cloned into a vector for the productionof an mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits, such as those provided by AmershamPharmacia Biotech, Promega (Madison Wis.), and US Biochemical. Suitablereporter molecules or labels which may be used for ease of detectioninclude radionuclides, enzymes, fluorescent, chemiluminescent, orchromogenic agents, as well as substrates, cofactors, inhibitors,magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding ASYNT may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or retained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeASYNT may be designed to contain signal sequences which direct secretionof ASYNT through a prokaryotic or eukaryotic cell membrane.

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a "prepro" form of theprotein may also be used to specify protein targeting, folding, and/oractivity. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and WI38), are available from the American TypeCulture Collection (ATCC, Nanassas Va.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding ASYNT may be ligated to aheterologous sequence resulting in translation of a fusion protein inany of the aforementioned host systems. For example, a chimeric ASYNTprotein containing a heterologous moiety that can be recognized by acommercially available antibody may facilitate the screening of peptidelibraries for inhibitors of ASYNT activity. Heterologous protein andpeptide moieties may also facilitate purification of fusion proteinsusing commercially available affinity matrices. Such moieties include,but are not limited to, glutathione S-transferase (GST), maltose bindingprotein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP),6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and6-His enable purification of their cognate fusion proteins onimmobilized glutathione, maltose, phenylarsine oxide, calmodulin, andmetal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA)enable immunoaffinity purification of fusion proteins using commerciallyavailable monoclonal and polyclonal antibodies that specificallyrecognize these epitope tags. A fusion protein may also be engineered tocontain a proteolytic cleavage site located between the ASYNT encodingsequence and the heterologous protein sequence, so that ASYNT may becleaved away from the heterologous moiety following purification.Methods for fusion protein expression and purification are discussed inAusubel (1995, supra, ch 10). A variety of commercially available kitsmay also be used to facilitate expression and purification of fusionproteins.

In a further embodiment of the invention, synthesis of radiolabeledASYNT may be achieved in vitro using the TNT rabbit reticulocyte lysateor wheat germ extract systems (Promega). These systems coupletranscription and translation of protein-coding sequences operablyassociated with the T7, T3, or SP6 promoters. Translation takes place inthe presence of a radiolabeled amino acid precursor, preferably ³⁵S-methionine.

Fragments of ASYNT may be produced not only by recombinant production,but also by direct peptide synthesis using solid-phase techniques. (See,e.g., Creighton, supra pp. 55-60.) Protein synthesis may be performed bymanual techniques or by automation. Automated synthesis may be achieved,for example, using the ABI 431A peptide synthesizer (Perkin-Elmer).Various fragments of ASYNT may be synthesized separately and thencombined to produce the full length molecule.

THERAPEUTICS

Chemical and structural similarity, e.g., in the context of sequencesand motifs, exists between regions of ASYNT and ATP synthase 6 subunit.In addition, the expression of ASYNT is closely associated withneurological tissues and cancer. Therefore, ASYNT appears to beassociated with neurodegenerative disorders, myopathies, cancers, andimmune disorders. In the treatment of disorders associated withincreased ASYNT activity, it is desirable to decrease the expression oractivity of ASYNT. In the treatment of disorders associated withdecreased ASYNT activity, it is desirable to increase the expression oractivity of ASYNT.

Therefore, in one embodiment, ASYNT or a fragment or derivative thereofmay be administered to a subject to treat or prevent a disorderassociated with decreased expression or activity of ASYNT. Examples ofsuch disorders include a cancer, such as adenocarcinoma, leukemia,lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, inparticular, cancers of the adrenal gland, bladder, bone, bone marrow,brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract,heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,prostate, salivary glands, skin, spleen, testis, thymus, thyroid, anduterus; a neurodegenerative disorder such as epilepsy, ischemiccerebrovascular disease, stroke, cerebral neoplasms, Alzheimer'sdisease, Pick's disease, Huntington's disease, dementia, Parkinson'sdisease and other extrapyramidal disorders, amyotrophic lateralsclerosis, and other motor neuron disorders, progressive neural muscularatrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosisand other demyelinating diseases, bacterial and viral meningitis, brainabscess, subdural empyema, epidural abscess, suppurative intracranialthrombophlebitis, myelitis and radiculitis, viral central nervous systemdisease; prion diseases including kuru, Creutzfeldt-Jakob disease, andGerstmann-Straussler-Scheinker syndrome; fatal familial insomnia,nutritional and metabolic diseases of the nervous system,neurofibromatosis, tuberous sclerosis, cerebelloretinalhemangioblastomatosis, encephalotrigeminal syndrome, Down's syndrome,mental retardation and other developmental disorders of the centralnervous system, Leigh syndrome, cerebral palsy, neuroskeletal disorders,autonomic nervous system disorders, cranial nerve disorders, spinal corddiseases, muscular dystrophy and other neuromuscular disorders,peripheral nervous system disorders, dermatomyositis and polymyositis;inherited, metabolic, endocrine, and toxic myopathies; myastheniagravis, periodic paralysis; mental disorders including mood, anxiety,and schizophrenic disorders; akathesia, amnesia, catatonia, diabeticneuropathy, tardive dyskinesia, dystonias, paranoid psychoses,postherpetic neuralgia, and Tourette's disorder; a myopathy, such ascardiomyopathy, encephalopathy, epilepsy, Keams-Sayre syndrome, lacticacidosis, myoclonic disorder, and ophthalmoplegia; and an immunedisorder such as acquired immunodeficiency syndrome (AIDS), Addison'sdisease, adult respiratory distress syndrome, allergies, ankylosingspondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmunehemolytic anemia, autoimmune thyroiditis, bronchitis, cholecystitis,contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, episodic lymphopenia withlymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophicgastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowelsyndrome, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupuserythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerativecolitis, uveitis, Wemer syndrome, complications of cancer, hemodialysis,and extracorporeal circulation, viral, bacterial, fungal, parasitic,protozoal, and helminthic infections, and trauma.

In another embodiment, a vector capable of expressing ASYNT or afragment or derivative thereof may be administered to a subject to treator prevent a disorder associated with decreased expression or activityof ASYNT including, but not limited to, those described above.

In a further embodiment, a pharmaceutical composition comprising asubstantially purified ASYNT in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent a disorder associated with-decreased expression or activity ofASYNT including, but not limited to, those provided above.

In still another embodiment, an agonist which modulates the activity ofASYNT may be administered to a subject to treat or prevent a disorderassociated with decreased expression or activity of ASYNT including, butnot limited to, those listed above.

In a further embodiment, an antagonist of ASYNT may be administered to asubject to treat or prevent a disorder associated with increasedexpression or activity of ASYNT. Such disorders may include, but are notlimited to, those discussed above. In one aspect, an antibody whichspecifically binds ASYNT may be used directly as an antagonist orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissue which express ASYNT.

In an additional embodiment, a vector expressing the complement of thepolynucleotide encoding ASYNT may be administered to a subject to treator prevent a disorder associated with increased expression or activityof ASYNT including, but not limited to, those described above.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences, or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of ASYNT may be produced using methods which are generallyknown in the art. In particular, purified ASYNT may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind ASYNT. Antibodies to ASYNT may also begenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric,and single chain antibodies, Fab fragments, and fragments produced by aFab expression library. Neutralizing antibodies (i.e., those whichinhibit dimer formation) are especially preferred for therapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans. and others may be immunized by injectionwith ASYNT or with any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium paryum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to ASYNT have an amino acid sequence consisting of atleast about 5 amino acids, and, more preferably, of at least about 10amino acids. It is also preferable that these oligopeptides, peptides,or fragments are identical to a portion of the amino acid sequence ofthe natural protein and contain the entire amino acid sequence of asmall, naturally occurring molecule. Short stretches of ASYNT aminoacids may be fused with those of another protein, such as KLH, andantibodies to the chimeric molecule may be produced.

Monoclonal antibodies to ASYNT may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120.)

In addition, techniques developed for the production of "chimericantibodies," such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (See, e.g., Morrison, S. L. et al.(1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al.(1984) Nature 312:604-608; and Takeda, S. et al. (1 985) Nature 314:452-454.) Alternatively, techniques described for the production ofsingle chain antibodies may be adapted, using methods known in the art,to produce ASYNT-specific single chain antibodies. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries. (See, e.g., Burton D. R. (1991) Proc. Natl. Acad. Sci.88:10134-10137.)

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature.(See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)

Antibody fragments which contain specific binding sites for ASYNT mayalso be generated. For example, such fragments include, but are notlimited to, F(ab')2 fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab')2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity. (See, e.g., Huse,W. D. et al. (1989) Science 246:1275-1281.)

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between ASYNT and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering ASYNT epitopes is preferred, but a competitivebinding assay may also be employed (Pound, supra).

Various methods such as Scatchard analysis in conjunction withradioimmunoassay techniques may be used to assess the affinity ofantibodies for ASYNT. Affinity is expressed as an association constant,K_(a), which is defined as the molar concentration of ASYNT-antibodycomplex divided by the molar concentrations of free antigen and freeantibody under equilibrium conditions. The K_(a) determined for apreparation of polyclonal antibodies, which are heterogeneous in theiraffinities for multiple ASYNT epitopes, represents the average affinity,or avidity, of the antibodies for ASYNT. The K_(a) determined for apreparation of monoclonal antibodies, which are monospecific for aparticular ASYNT epitope, represents a true measure of affinity.High-affinity antibody preparations with K_(a) ranging from about 10⁹ to10¹² l/mole are preferred for use in immunoassays in which theASYNT-antibody complex must withstand rigorous manipulations.Low-affinity antibody preparations with K_(a) ranging from about 10⁶ to10⁷ l/mole are preferred for use in immunopurification and similarprocedures which ultimately require dissociation of ASYNT, preferably inactive form, from the antibody (Catty, D. (1988) Antibodies, Volume I; APractical Approach, IRL Press, Washington D.C.; Liddell, J. E. andCryer, A. (1991) A Practical Guide to Monoclonal Antibodies, John Wiley& Sons, New York, N.Y.).

The titer and avidity of polyclonal antibody preparations may be furtherevaluated to determine the quality and suitability of such preparationsfor certain downstream applications. For example, a polyclonal antibodypreparation containing at least 1-2 mg specific antibody/ml, preferably5-10 mg specific antibody/ml, is preferred for use in proceduresrequiring precipitation of ASYNT-antibody complexes. Procedures forevaluating antibody specificity, titer, and avidity, and guidelines forantibody quality and usage in various applications, are generallyavailable. (See, e.g., Catty, supra, and Coligan et al. supra.)

In another embodiment of the invention, the polynucleotides encodingASYNT, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding ASYNT may be used in situations in which itwould be desirable to block the transcription of the mRNA. Inparticular, cells may be transformed with sequences complementary topolynucleotides encoding ASYNT. Thus, complementary molecules orfragments may be used to modulate ASYNT activity, or to achieveregulation of gene function. Such technology is now well known in theart, and sense or antisense oligonucleotides or larger fragments can bedesigned from various locations along the coding or control regions ofsequences encoding ASYNT.

Expression vectors derived from retroviruses, adenoviruses, or herpes orvaccinia viruses, or from various bacterial plasmids, may be used fordelivery of nucleotide sequences to the targeted organ, tissue, or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors to express nucleic acid sequencescomplementary to the polynucleotides encoding ASYNT. (See, e.g.,Sambrook, supra; Ausubel, 1995, supra.)

Genes encoding ASYNT can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide,or fragment thereof, encoding ASYNT. Such constructs may be used tointroduce untranslatable sense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector, and may last even longer if appropriatereplication elements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning complementary sequences or antisense molecules (DNA, RNA, orPNA) to the control, 5', or regulatory regions of the gene encodingASYNT. Oligonucleotides derived from the transcription initiation site,e.g., between about positions -10 and +10 from the start site, arepreferred. Similarly, inhibition can be achieved using triple helixbase-pairing methodology. Triple helix pairing is useful because itcauses inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature. (See, e.g., Gee, J. E. et al. (1994)in Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches,Futura Publishing, Mt. Kisco, N.Y., pp. 163-177.) A complementarysequence or antisense molecule may also be designed to block translationof mRNA by preventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of sequences encodingASYNT.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding ASYNT. SuchDNA sequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA, constitutivelyor inducibly, can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5' and/or 3' ends of the molecule,or the use of phosphorothioate or 2' O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections, or bypolycationic amino polymers may be achieved using methods which are wellknown in the art. (See, e.g., Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-466.)

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical or sterile composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of ASYNT,antibodies to ASYNT, and mimetics, agonists, antagonists, or inhibitorsof ASYNT. The compositions may be administered alone or in combinationwith at least one other agent, such as a stabilizing compound, which maybe administered in any sterile, biocompatible pharmaceutical carrierincluding, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs, or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing, Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with fillers or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils, such as sesame oil, or synthetic fatty acid esters, such asethyl oleate, triglycerides, or liposomes. Non-lipid polycationic aminopolymers may also be used for delivery. Optionally, the suspension mayalso contain suitable stabilizers or agents to increase the solubilityof the compounds and allow for the preparation of highly concentratedsolutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tendto be more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7%mannitol, at a pH range of 4.5 to 5.5, that is combined with bufferprior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of ASYNT, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells or inanimal models such as mice, rats, rabbits, dogs, or pigs. An animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example ASYNT or fragments thereof, antibodies of ASYNT,and agonists, antagonists or inhibitors of ASYNT, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED₅₀ (the dosetherapeutically effective in 50% of the population) or LD₅₀ (the doselethal to 50% of the population) statistics. The dose ratio of toxic totherapeutic effects is the therapeutic index, which can be expressed asthe LD₅₀ /ED₅₀ ratio. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies are used to formulate a range of dosage forhuman use. The dosage contained in such compositions is preferablywithin a range of circulating concentrations that includes the ED₅₀ withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, the sensitivity of the patient, and theroute of administration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting pharmaceuticalcompositions may be administered every 3 to 4 days, every week, orbiweekly depending on the half-life and clearance rate of the particularformulation.

Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to atotal dose of about 1 gram, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

DIAGNOSTICS

In another embodiment, antibodies which specifically bind ASYNT may beused for the diagnosis of neurodegenerative disorders, myopathies,cancer, and immune disorders characterized by expression of ASYNT, or inassays to monitor patients being treated with ASYNT or agonists,antagonists, or inhibitors of ASYNT. Antibodies useful for diagnosticpurposes may be prepared in the same manner as described above fortherapeutics. Diagnostic assays for ASYNT include methods which utilizethe antibody and a label to detect ASYNT in human body fluids or inextracts of cells or tissues. The antibodies may be used with or withoutmodification, and may be labeled by covalent or non-covalent attachmentof a reporter molecule. A wide variety of reporter molecules, several ofwhich are described above, are known in the art and may be used.

A variety of protocols for measuring ASYNT, including ELISAs, RIAs, andFACS, are known in the art and provide a basis for diagnosing altered orabnormal levels of ASYNT expression. Normal or standard values for ASYNTexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toASYNT under conditions suitable for complex formation. The amount ofstandard complex formation may be quantitated by various methods,preferably by photometric means. Quantities of ASYNT expressed insubject, samples from biopsied tissues are compared with the standardvalues. Deviation between standard and subject values establishes theparameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingASYNT may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofASYNT may be correlated with disease. The diagnostic assay may be usedto determine absence, presence, and excess expression of ASYNT, and tomonitor regulation of ASYNT levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding ASYNT or closely related molecules may be used to identifynucleic acid sequences which encode ASYNT. The specificity of the probe,whether it is made from a highly specific region, e.g., the 5'regulatory region, or from a less specific region, e.g., a conservedmotif, and the stringency of the hybridization or amplification(maximal, high, intermediate, or low), will determine whether the probeidentifies only naturally occurring sequences encoding ASYNT, allelicvariants, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably have at least 50% sequence identity to any of theASYNT encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and may be derived from the sequence of SEQID NO:2 or from genomic sequences including promoters, enhancers, andintrons of the ASYNT gene.

Means for producing specific hybridization probes for DNAs encodingASYNT include the cloning of polynucleotide sequences encoding ASYNT orASYNT derivatives into vectors for the production of mRNA probes. Suchvectors are known in the art, are commercially available, and may beused to synthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³² p or ³⁵ S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

Polynucleotide sequences encoding ASYNT may be used for the diagnosis ofneurodegenerative disorders, myopathies, cancers, and immune disordersassociated with expression of ASYNT Examples of such disorders include,but are not limited to, a cancer, such as adenocarcinoma, leukemia,lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, inparticular, cancers of the adrenal,gland, bladder, bone, bone marrow.brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract,heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,prostate, salivary glands, skin, spleen, testis, thymus, thyroid, anduterus; a neurodegenerative disorder such as epilepsy, ischemiccerebrovascular disease, stroke, cerebral neoplasms, Alzheimer'sdisease, Pick's disease, Huntington's disease, dementia, Parkinson'sdisease and other extrapyramidal disorders, amyotrophic lateralsclerosis, and other motor neuron disorders, progressive neural muscularatrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosisand other demyelinating diseases, bacterial and viral meningitis, brainabscess, subdural empyema, epidural abscess, suppurative intracranialthrombophlebitis, myelitis and radiculitis, viral central nervous systemdisease; prion diseases including kuru, Creutzfeldt-Jakob disease, andGerstmann-Straussler-Scheinker syndrome; fatal familial insomnia,nutritional and metabolic diseases of the nervous system,neurofibromatosis, tuberous sclerosis, cerebelloretinalhemangioblastomatosis, encephalotrigeminal syndrome, Down's syndrome,mental retardation and other developmental disorders of the centralnervous system, Leigh syndrome, cerebral palsy, neuroskeletal disorders,autonomic nervous system disorders, cranial nerve disorders, spinal corddiseases, muscular dystrophy and other neuromuscular disorders,peripheral nervous system disorders, dermatomyositis and polymyositis;inherited, metabolic, endocrine, and toxic myopathies; myastheniagravis, periodic paralysis; mental disorders including mood, anxiety,and schizophrenic disorders; akathesia, amnesia, catatonia, diabeticneuropathy, tardive dyskinesia, dystonias, paranoid psychoses,postherpetic neuralgia, and Tourette's disorder; a myopathy, such ascardiomyopathy, encephalopathy, epilepsy, Kearns-Sayre syndrome, lacticacidosis, myoclonic disorder, and ophthalmoplegia; and an immunedisorder such as acquired immunodeficiency syndrome (AIDS), Addison'sdisease, adult respiratory distress syndrome, allergies, ankylosingspondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmunehemolytic anemia, autoimmune thyroiditis, bronchitis, cholecystitis,contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, episodic lymphopenia withlymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophicgastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowelsyndrome, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupuserythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerativecolitis, uveitis, Werner syndrome, complications of cancer,hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,parasitic, protozoal, and helminthic infections and trauma. Thepolynucleotide sequences encoding ASYNT may be used in Southern ornorthern analysis, dot blot, or other membrane-based technologies; inPCR technologies; in dipstick, pin, and multiformat ELISA-like assays;and in microarrays utilizing fluids or tissues from patients to detectaltered ASYNT expression. Such qualitative or quantitative methods arewell known in the art.

In a particular aspect, the nucleotide sequences encoding ASYNT may beuseful in assays that detect the presence of associated disorders,particularly those mentioned above. The nucleotide sequences encodingASYNT may be labeled by standard methods and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the patient sample is significantlyaltered in comparison to a control sample then the presence of alteredlevels of nucleotide sequences encoding ASYNT in the sample indicatesthe presence of the associated disorder. Such assays may also be used toevaluate the efficacy of a particular therapeutic treatment regimen inanimal studies, in clinical trials, or to monitor the treatment of anindividual patient.

In order to provide a basis for the diagnosis of a disorder associatedwith expression of ASYNT, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, encoding ASYNT, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withvalues from an experiment in which a known amount of a substantiallypurified polynucleotide is used. Standard values obtained in this mannermay be compared with values obtained from samples from patients who aresymptomatic for a disorder. Deviation from standard values is used toestablish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocolis initiated, hybridization assays may be repeated on a regular basis todetermine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

With respect to cancer, the presence of an abnormal amount of transcript(either under- or over-expressed) in biopsied tissue from an individualmay indicate a predisposition for the development of the disease, or mayprovide a means for detecting the disease prior to the appearance ofactual clinical symptoms. A more definitive diagnosis of this type mayallow health professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding ASYNT may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced in vitroOligomers will preferably contain a fragment of a polynucleotideencoding ASYNT, or a fragment of a polynucleotide complementary to thepolynucleotide encoding ASYNT, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantitation of closely related DNA or RNA sequences.

Methods which may also be used to quantitate the expression of ASYNTinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and interpolating results from standard curves.(See, e.g., Melby, P. C. et al. (1993) J. Immunol. Methods 159:235-244;Duplaa, C. et al. (1993) Anal. Biochem. 212-229.) The speed ofquantitation of multiple samples may be accelerated by running the assayin an ELISA format where the oligomer of interest is presented invarious dilutions and a spectrophotometric or colorimetric responsegives rapid quantitation.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotide sequences described herein may be used astargets in a microarray. The microarray can be used to monitor theexpression level of large numbers of genes simultaneously and toidentify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, and to develop andmonitor the activities of therapeutic agents.

Microarrays may be prepared, used, and analyzed using methods known inthe art. (See, e.g., Brennan, T. M. et al. (1995) U.S. Pat. No.5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci.93:10614-10619; Baldeschweiler et al. (1995) PCT applicationWO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505;Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155; andHeller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)

In another embodiment of the invention, nucleic acid sequences encodingASYNT may be used to generate hybridization probes useful in mapping thenaturally occurring genomic sequence. The sequences may be mapped to aparticular chromosome, to a specific region of a chromosome, or toartificial chromosome constructions, e.g., human artificial chromosomes(HACs), yeast artificial chromosomes (YACs), bacterial artificialchromosomes (BACs), bacterial P1 constructions, or single chromosomecDNA libraries. (See, e.g., Harrington, J. J. et al. (1997) Nat Genet.15:345-355; Price, C. M. (1993) Blood Rev. 7:127-134; and Trask, B. J.(1991) Trends Genet. 7:149-154.)

Fluorescent in situ hybridization (FISH) may be correlated with otherphysical chromosome mapping techniques and genetic map data. (See, e.g.,Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968.) Examples ofgenetic map data can be found in various scientific journals or at theOnline Mendelian Inheritance in Man (OMIM) site. Correlation between thelocation of the gene encoding ASYNT on a physical chromosomal map and aspecific disorder, or a predisposition to a specific disorder, may helpdefine the region of DNA associated with that disorder. The nucleotidesequences of the invention may be used to detect differences in genesequences among normal, carrier, and affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques, such as linkage analysis using established chromosomalmarkers, may be used for extending genetic maps. Often the placement ofa gene on the chromosome of another mammalian species, such as mouse,may reveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms by physical mapping. This provides valuable informationto investigators searching for disease genes using positional cloning orother gene discovery techniques. Once the disease or syndrome has beencrudely localized by genetic linkage to a particular genomic region,e.g., ataxia-telangiectasia to 11 q22-23, any sequences mapping to thatarea may represent associated or regulatory genes for furtherinvestigation. (See, e.g., Gatti, R. A. et al. (1988) Nature336:577-580.) The nucleotide sequence of the subject invention may alsobe used to detect differences in the chromosomal location due totranslocation, inversion, etc., among normal, carrier, or affectedindividuals.

In another embodiment of the invention, ASYNT, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes betweenASYNT and the agent being tested may be measured.

Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest. (See, e.g., Geysen, et al. (1984) PCT applicationWO84/03564.) In this method, large numbers of different small testcompounds are synthesized on a solid substrate. The test compounds arereacted with ASYNT, or fragments thereof, and washed. Bound ASYNT isthen detected by methods well known in the art. Purified ASYNT can alsobe coated directly onto plates for use in the aforementioned drugscreening techniques. Alternatively, non-neutralizing antibodies can beused to capture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding ASYNT specificallycompete with a test compound for binding ASYNT. In this manner,antibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with ASYNT.

In additional embodiments, the nucleotide sequences which encode ASYNTmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES

I. Construction of cDNA Libraries

The BLADTUT07 cDNA library was constructed from tumorous bladder tissueobtained from a 58-year-old Caucasian male during a radical cystectomy.The patient was diagnosed with grade 3 transitional cell carcinomaforming an ulcerated, infiltrative mass situated in the left lateralbladder wall. The tumor extended through the muscularis into perivesicalfat. The remaining bladder showed marked cystitis with scatteredmicroscopic foci of transitional cell carcinoma in situ. The familyhistory included acute myocardial infarction, atherosclerosis, anddiabetes in a sibling.

Frozen tissue was homogenized and lysed in guanidinium isothiocyanatesolution using a Polytron PT-3000 homogenizer (Brinkmann Instruments,Westbury, N.Y.). The lysate was centrifuged over a CsCl cushion toisolate RNA. The RNA was extracted with acid phenol, precipitated withsodium acetate and ethanol, resuspended in RNase-free water, and treatedwith DNase. The RNA was re-extracted with acid phenol and reprecipitatedwith sodium acetate and ethanol. Poly(A+) RNA was isolated using theOLIGOTEX mRNA purification kit (QIAGEN, Chatsworth, Calif.).

Poly(A+) RNA was used for cDNA synthesis and construction of the cDNAlibrary according to the recommended protocols in the SUPERSCRIPTplasmid system (Life Technologies). The cDNAs were fractionated on aSEPHAROSE CL4B column (Amersham Pharmacia Biotech), and those cDNAsexceeding 400 bp were ligated into pINCY (Incyte Pharmaceuticals).Recombinant plasmids were transformed into DH5α competent cells (LifeTechnologies).

II. Isolation of cDNA Clones

Plasmid DNA was released from the cells and purified using the R.E.A.L.Prep 96 plasmid kit (QIAGEN). The recommended protocol was employedexcept for the following changes: 1) the bacteria were cultured in 1 mlof sterile Terrific Broth (Life Technologies) with carbenicillin at 25mg/l and glycerol at 0.4%; 2) after the cultures were incubated for 19hours, the cells were lysed with 0.3 ml of lysis buffer; and 3)following isopropanol precipitation, the plasmid DNA pellets were eachresuspended in 0.1 ml of distilled water. The DNA samples were stored at4° C.

III. Sequencing and Analysis

The cDNAs were prepared for sequencing using the ABI CATALYST 800(Perkin-Elmer) or the HYDRA microdispenser (Robbins Scientific) orMICROLAB 2200 (Hamilton) liquid tranfer systems in combination with thePTC-200 thermal cyclers (MJ Research). The cDNAs were sequenced usingthe ABI PRISM 373 or 377 sequencing systems (Perkin-Elmer) and standardABI protocols, base calling software, and kits. In one alternative,cDNAs were sequenced using the MEGABACE 1000 DNA sequencing system(Molecular Dynamics). In another alternative, the cDNAs were amplifiedand sequenced using the ABI PRISM BIGDYE Terminator cycle sequencingready reaction kit (Perkin-Elmer). In yet another alternative, cDNAswere sequenced using solutions and dyes from Amersham Pharmacia Biotech.Reading frames for the ESTs were determined using standard methods(reviewed in Ausubel, 1997, supra, unit 7.7). Some of the cDNA sequenceswere selected for extension using the techniques disclosed in Example V.

The polynucleotide sequences derived from cDNA, extension, and shotgunsequencing were assembled and analyzed using a combination of softwareprograms which utilize algorithms well known to those skilled in theart. Table 1 summarizes the software programs, descriptions, references,and threshold parameters used. The first column of Table 1 shows thetools, programs, and algorithms used, the second column provides a briefdescription thereof, the third column presents the references which areincorporated by reference herein, and the fourth column presents, whereapplicable, the scores, probability values, and other parameters used toevaluate the strength of a match between two sequences (the higher theprobability the greater the homology). Sequences were analyzed usingMACDNASIS PRO software (Hitachi Software Engineering) and LASERGENEsoftware (DNASTAR).

The polynucleotide sequences were validated by removing vector, linker,and polyA sequences and by masking ambiguous bases, using algorithms andprograms based on BLAST, dynamic programming, and dinucleotide nearestneighbor analysis. The sequences were then queried against a selectionof public databases such as GenBank primate, rodent, mammalian,vertebrate, and eukaryote databases, and BLOCKS to acquire annotation,using programs based on BLAST, FASTA, and BLIMPS. The sequences wereassembled into full length polynucleotide sequences using programs basedon Phred, Phrap, and Consed, and were screened for open reading framesusing programs based on GeneMark, BLAST, and FASTA. The full lengthpolynucleotide sequences were translated to derive the correspondingfull length amino acid sequences, and these full length sequences weresubsequently analyzed by querying against databases such as the GenBankdatabases (described above), SwissProt, BLOCKS, PRINTS, PFAM, andProsite.

The programs described above for the assembly and analysis of fulllength polynucleotide and amino acid sequences were used to identifypolynucleotide sequence fragments from SEQ ID NO:2. Fragments from about20 to about 4000 nucleotides which are useful in hybridization andamplification technologies were described in The Invention sectionabove.

IV. Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7;Ausubel, 1995, supra, ch. 4 and 16.)

Analogous computer techniques applying BLAST were used to search foridentical or related molecules in nucleotide databases such as GenBankor LIFESEQ database (Incyte Pharmaceuticals, Palo Alto, Calif.). Thisanalysis is much faster than multiple membrane-based hybridizations. Inaddition, the sensitivity of the computer search can be modified todetermine whether any particular match is categorized as exact orsimilar. The basis of the search is the product score, which is definedas:

    % sequence identitv×% maximum BLAST score

The product score takes into account both the degree of similaritybetween two sequences and the length of the sequence match. For example,with a product score of 40, the match will be exact within a 1% to 2%error, and, with a product score of 70, the match will be exact. Similarmolecules are usually identified by selecting those which show productscores between 15 and 40, although lower scores may identify, relatedmolecules.

The results of northern analyses are reported a percentage distributionof libraries in which the transcript encoding ASYNT occurred. Analysisinvolved the categorization of cDNA libraries by organ/tissue anddisease. The organ/tissue categories included cardiovascular,dermatologic, developmental, endocrine, gastrointestinal,hematopoietic/immune, musculoskeletal, nervous, reproductive, andurologic. The disease categories included cancer, inflammation/trauma,fetal, neurological, and pooled. For each category, the number oflibraries expressing the sequence of interest was counted and divided bythe total number of libraries across all categories. Percentage valuesof tissue-specific and disease expression are reported in thedescription of the invention.

V. Extension of ASYNT Encoding Polynucleotides

The full length nucleic acid sequence of SEQ ID NO:2 was produced byextension of an appropriate fragment of the full length molecule usingoligonucleotide primers designed from this fragment. One primer wassynthesized to initiate 5' extension of the known fragment, and theother primer, to initiate 3' extension of the known fragment. Theinitial primers were designed using OLIGO 4.06 primer analysis software(National Biosciences), or another appropriate program, to be about 22to 30 nucleotides in length, to have a GC content of about 50% or more,and to anneal to the target sequence at temperatures of about 68° C. toabout 72° C. Any stretch of nucleotides which would result in hairpinstructures and primer-primer dimerizations was avoided.

Selected human cDNA libraries were used to extend the sequence. If morethan one extension was necessary or desired, additional or nested setsof primers were designed.

High fidelity amplification was obtained by PCR using methods well knownin the art. PCR was performed in 96-well plates using the PTC-200thermal cycler (MJ Research, Inc.). The reaction mix contained DNAtemplate, 200 nmol of each primer, reaction buffer containing Mg² +,(NH₄)₂ SO₄, and β-mercaptoethanol, Taq DNA polymerase (AmershamPharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNApolymerase (Stratagene), with the following parameters for primer pairPCI A and PCI B: Step 1: 94° C., 3 min; Step 2: 94° C., 15 sec; Step 3:60° C., 1 min; Step 4: 68 5: Steps 2, 3, and 4 repeated 20 times; Step6: 68° C., 5 min; Step 7: storage at 4° C. In the alternative, theparameters for primer pair T7 and SK+were as follows: Step 1: 94° C., 3min; Step 2: 94° C., 5 sec 3: 57° C., 1 min; Step 4: 68° C., 2 min; Step5: Steps 2, 3, and 4 repeated 20 times; Step Step 7: storage at 4° C.

The concentration of DNA in each well was determined by dispensing 100μl PICO GREEN quantitation reagent (0.25% (v/v) PICO GREEN; MolecularProbes, Eugene OR) dissolved in 1 X TE and 0.5 μl of undiluted PCRproduct into each well of an opaque fluorimeter plate (Coming Costar,Acton, Mass.), allowing the DNA to bind to the reagent. The plate wasscanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measurethe fluorescence of the sample and to quantify the concentration of DNA.A 5 μl to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a 1% agarose mini-gel to determine which reactionswere successful in extending the sequence.

The extended nucleotides were desalted and concentrated, transferred to384-well plates, digested with CviJI cholera virus endonuclease(Molecular Biology Research, Madison, Wis.), and sonicated or shearedprior to religation into pUC 18 vector (Amersham Pharmacia Biotech). Forshotgun sequencing, the digested nucleotides were separated on lowconcentration (0.6 to 0.8%) agarose gels, fragments were excised, andagar digested with Agar ACE (Promega). Extended clones were religatedusing T4 ligase (New England Biolabs, Beverly, Mass.) into pUC 18 vector(Amersham Pharmacia Biotech), treated with Pfu DNA polymerase(Stratagene) to fill-in restriction site overhangs, and transfected intocompetent E. coli cells. Transformed cells were selected onantibiotic-containing media, individual colonies were picked andcultured overnight at 37° C. in 384-well plates in LB/2× carb liquidmedia.

The cells were lysed, and DNA was amplified by PCR using Taq DNApolymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase(Stratagene) with the following parameters: Step 1: 94° C., 3 min; Step2: 94° C., 15 sec; Step 3: 60° C., 1 min; Step 4: 72° C., 2 min; Step 5:steps 2, 3, and 4 repeated 29 times; Step 6: 72° C., 5 min; Step 7:storage at 4° C. DNA was quantified by PICOGREEN reagent (MolecularProbes) as described above. Samples with low DNA recoveries werereamplified using the same conditions as described above. Samples werediluted with 20% dimethysulphoxide (1:2, v/v), and sequenced usingDYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit(Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator cyclesequencing ready reaction kit (Perkin-Elmer).

In like manner, the nucleotide sequence of SEQ ID NO:2 is used to obtain5' regulatory sequences using the procedure above, oligonucleotidesdesigned for such extension, and an appropriate genomic library.

VI. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 are employed to screencDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 software (National Biosciences) and labeled bycombining 50 pmol of each oligomer, 250 μCi of [³² P]-adenosinetriphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase(DuPont NEN, Boston, Mass.). The labeled oligonucleotides aresubstantially purified using a SEPHADEX G-25 superfine size exclusiondextran bead column (Amersham Pharmacia Biotech). An aliquot containing10⁷ counts per minute of the labeled probe is used in a typicalmembrane-based hybridization analysis of human genomic DNA digested withone of the following endonucleases: Ase I. Bgl II, Eco RI, Pst I, XbaI,or Pvu II (DuPont NEN).

The DNA from each digest is fractionated on a 0.7% agarose gel andtransferred to nylon membranes (NYTRAN PLUS, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1× salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT-AR film(Eastman Kodak, Rochester, N.Y.) is exposed to the blots, hybridizationpatterns are compared visually.

VII. Microarrays

A chemical coupling procedure and an ink jet device can be used tosynthesize array elements on the surface of a substrate. (See, e.g.,Baldeschweiler, supra.) An array analogous to a dot or slot blot mayalso be used to arrange and link elements to the surface of a substrateusing thermal, UV, chemical, or mechanical bonding procedures. A typicalarray may be produced by hand or using available methods and machinesand contain any appropriate number of elements. After hybridization,nonhybridized probes are removed and a scanner used to determine thelevels and patterns of fluorescence. The degree of complementarity andthe relative abundance of each probe which hybridizes to an element onthe microarray may be assessed through analysis of the scanned images.

Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereofmay comprise the elements of the microarray. Fragments suitable forhybridization can be selected using software well known in the art suchas LASERGENE software (DNASTAR). Full-length cDNAs, ESTs, or fragmentsthereof corresponding to one of the nucleotide sequences of the presentinvention, or selected at random from a cDNA library relevant to thepresent invention, are arranged on an appropriate substrate, e.g., aglass slide. The cDNA is fixed to the slide using, e.g., UVcross-linking followed by thermal and chemical treatments and subsequentdrying. (See, e.g., Schena, M. et al. (1995) Science 270:467-470;Shalon, D. et al. (1996) Genome Res. 6:639-645.) Fluorescent probes areprepared and used for hybridization to the elements on the substrate.The substrate is analyzed by procedures described above.

VIII. Complementary Polynucleotides

Sequences complementary to the ASYNT-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring ASYNT. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO 4.06 primeranalysis software (National Biosciences) and the coding sequence ofASYNT. To inhibit transcription, a complementary oligonucleotide isdesigned from the most unique 5' sequence and used to prevent promoterbinding to the coding sequence. To inhibit translation, a complementaryoligonucleotide is designed to prevent ribosomal binding to theASYNT-encoding transcript.

IX. Expression of ASYNT

Expression and purification of ASYNT is achieved using bacterial orvirus-based expression systems. For expression of ASYNT in bacteria,cDNA is subcloned into an appropriate vector containing an antibioticresistance gene and an inducible promoter that directs high levels ofcDNA transcription. Examples of such promoters include, but are notlimited to, the trp-lac (tac) hybrid promoter and the T5 or T7bacteriophage promoter in conjunction with the lac operator regulatoryelement. Recombinant vectors are transformed into suitable bacterialhosts, e.g., BL21(DE3). Antibiotic resistant bacteria express ASYNT uponinduction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expressionof ASYNT in eukaryotic cells is achieved by infecting insect ormammalian cell lines with recombinant Autographica californica nuclearpolyhedrosis virus (AcMNPV), commonly known as baculovirus. Thenonessential polyhedrin gene of baculovirus is replaced with cDNAencoding ASYNT by either homologous recombination or bacterial-mediatedtransposition involving transfer plasmid intermediates. Viralinfectivity is maintained and the strong polyhedrin promoter drives highlevels of cDNA transcription. Recombinant baculovirus is used to infectSpodoptera frugiperda (Sf9) insect cells in most cases, or humanhepatocytes, in some cases. Infection of the latter requires additionalgenetic modifications to baculovirus. (See Engelhard, E. K. et al.(1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)Hum. Gene Ther. 7:1937-1945.)

In most expression systems, ASYNT is synthesized as a fusion proteinwith, e.g., glutathione S-transferase (GST) or a peptide epitope tag,such as FLAG or 6-His, permitting rapid, single-step, affinity-basedpurification of recombinant fusion protein from crude cell lysates. GST,a 26-kilodalton enzyme from Schistosoma japonicum, enables thepurification of fusion proteins on immobilized glutathione underconditions that maintain protein activity and antigenicity (AmershamPharmacia Biotech). Following purification, the GST moiety can beproteolytically cleaved from ASYNT at specifically engineered sites.FLAG, an 8-amino acid peptide, enables immunoaffinity purification usingcommercially available monoclonal and polyclonal anti-FLAG antibodies(Eastman Kodak). 6-His, a stretch of six consecutive histidine residues,enables purification on metal-chelate resins (QIAGEN). Methods forprotein expression and purification are discussed in Ausubel (1995,supra, ch 10 and 16). Purified ASYNT obtained by these methods can beused directly in the following activity assay.

X. Demonstration of ASYNT Activity

The activity of ASYNT in a functional F_(o) portion of the ATP synthasemolecule is based on the fact that ATP synthesis from ADP and inorganicphosphate can be measured when F_(o) is reconstituted with the F₁ ATPaseand incubated together with a submitochondrial particle fractionprepared from bovine mitochondria which provides a source of electrontransport from NADH to O₂. ASYNT is first incorporated into areconstituted F₀ molecule, and this molecule is further reconstitutedwith F₁ ATPase to form a functional ATP synthase. Bovinesubmitochondrial particles are then prepared by sonication of intactmitochondria and isolated from the preparation by differentialcentrifugation. The assay is performed by incubating the reconstitutedATP synthase, submitochondrial particles, NADH, ADP, and P³² together ina suitable buffer, The reaction product, ATP³², is separated fromunreacted P³² by electrophoresis and quantitated using a gammaradioisotope counter. The amount of ATP³² recovered is proportional tothe activity of ASYNT.

XI. Functional Assays

ASYNT function is assessed by expressing the sequences encoding ASYNT atphysiologically elevated levels in mammalian cell culture systems. cDNAis subcloned into a mammalian expression vector containing a strongpromoter that drives high levels of cDNA expression. Vectors of choiceinclude pCMV SPORT plasmi (Life Technologies) and pCR3.1 plasmi(Invitrogen, Carlsbad, Calif.), both of which contain thecytomegalovirus promoter. 5-10 μg of recombinant vector are transientlytransfected into a human cell line, preferably of endothelial orhematopoietic origin, using either liposome formulations orelectroporation. 1-2 μg of an additional plasmid containing sequencesencoding a marker protein are co-transfected. Expression of a markerprotein provides a means to distinguish transfected cells fromnontransfected cells and is a reliable predictor of cDNA expression fromthe recombinant vector. Marker proteins of choice include, e.g., GreenFluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein.Flow cytometry (FCM), an automated, laser optics-based technique, isused to identify transfected cells expressing GFP or CD64-GFP, and toevaluate cellular properties, for example, their apoptotic state. FCMdetects and quantifies the uptake of fluorescent molecules that diagnoseevents preceding or coincident with cell death. These events includechanges in nuclear DNA content as measured by staining of DNA withpropidium iodide; changes in cell size and granularity as measured byforward light scatter and 90 degree side light scatter; down-regulationof DNA synthesis as measured by decrease in bromodeoxyuridine uptake;alterations in expression of cell surface and intracellular proteins asmeasured by reactivity with specific antibodies; and alterations inplasma membrane composition as measured by the binding offluorescein-conjugated Annexin V protein to the cell surface. Methods inflow cytometry are discussed in Ormerod, M. G. (1 994) Flow Cytometry,Oxford, New York, N.Y.

The influence of ASYNT on gene expression can be assessed using highlypurified populations of cells transfected with sequences encoding ASYNTand either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on thesurface of transfected cells and bind to conserved regions of humanimmunoglobulin G (IgG). Transfected cells are efficiently separated fromnontransfected cells using magnetic beads coated with either human IgGor antibody against CD64 (DYNAL, Lake Success, N.Y.). mRNA can bepurified from the cells using methods well known by those of skill inthe art. Expression of mRNA encoding ASYNT and other genes of interestcan be analyzed by northern analysis or microarray techniques.

XII. Production of ASYNT Specific Antibodies

ASYNT substantially purified using polyacrylamide gel electrophoresis(PAGE; see, e.g., Harrington, M. G. (1990) Methods Enzymol.182:488-495), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols.

Alternatively, the ASYNT amino acid sequence is analyzed using LASERGENEsoftware (DNASTAR) to determine regions of high immunogenicity, and acorresponding oligopeptide is synthesized and used to raise antibodiesby means known to those of skill in the art. Methods for selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions are well described in the art. (See, e.g., Ausubel,1995, supra, ch. 11.)

Typically, oligopeptides 15 residues in length are synthesized using anABI 431A peptide synthesizer (Perkin-Elmer) using fmoc-chemistry andcoupled to KLH (Sigma-Aldrich, St. Louis, Mo.) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increaseimmunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunizedwith the oligopeptide-KLH complex in complete Freund's adjuvant.Resulting antisera are tested for antipeptide activity by, for example,binding the peptide to plastic, blocking with 1% BSA, reacting withrabbit antisera, washing, and reacting with radio-iodinated goatanti-rabbit IgG.

XIII. Purification of Naturally Occurring ASYNT Using SpecificAntibodies

Naturally occurring or recombinant ASYNT is substantially purified byimmunoaffinity chromatography using antibodies specific for ASYNT. Animmunoaffinity column is constructed by covalently coupling anti-ASYNTantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin isblocked and washed according to the manufacturer's instructions.

Media containing ASYNT are passed over the immunoaffinity column, andthe column is washed under conditions that allow the preferentialabsorbance of ASYNT (e.g., high ionic strength buffers in the presenceof detergent). The column is eluted under conditions that disruptantibody/ASYNT binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), andASYNT is collected.

XIV. Identification of Molecules Which Interact with ASYNT

ASYNT, or biologically active fragments thereof, are labeled with ¹²⁵ IBolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J.133:529.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled ASYNT, washed, and anywells with labeled ASYNT complex are assayed. Data obtained usingdifferent concentrations of ASYNT are used to calculate values for thenumber, affinity, and association of ASYNT with the candidate molecules.Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

                                      TABLE 1                                     __________________________________________________________________________    Program Description              Reference           Parameter Threshold      __________________________________________________________________________    ABI FACTURA                                                                           A program that removes vector sequences and masks                                                      Perkin-Elmer Applied Biosystems,                                                                    ambiguous bases in                                                          nucleic acid                                                                  sequences. Foster                                                             City, CA.                  ABI/PARACEL A Fast Data Finder useful in comparing and annotating                                                                Perkin-Elmer Applied                                                          Biosystems, Mismatch                                                          <50%                       FDF amino acid or nucleic acid sequences. Foster City, CA; Paracel                                                               Inc., Pasadena, CA.                                                            ABI A program that                                                           assembles nucleic                                                             acid sequences.                                                               Perkin-Elmer Applied                                                          Biosystems,                AutoAssembler  Foster City, CA.                                               BLAST A Basic Local Alignment Search Tool useful in sequence Altschul,                                                           S. F. et al., (1990)                                                          J. Mol. Biol. ESTs:                                                           Probability                 similarity search for amino acid and nucleic acid sequences. 215:                                                               403-410; Altschul,                                                            S. F. et al. (1997)                                                           value = 1.0E-8 or                                                             less                        BLAST includes five functions: blastp, blastn, blastx, Nucleic Acids                                                            Res. 25: 3389-3402.                                                           Full Length sequences                                                         :                           tblastn, and tblastx.  Probability value =                                      1.0E-10 or less                                                            FASTA A Pearson and Lipman algorithm that searches for Pearson, W. R.                                                            and D. J. Lipman                                                              (1988) Proc. ESTs:                                                            fasta E value =                                                                 similarity between                                                          a query sequence and                                                          a group of Natl.                                                              Acad Sci. 85:                                                                 2444-2448; Pearson,                                                           W. R. 1.06E-6                                                                 Assembled                   sequences of the same type. FASTA comprises as least five (1990)                                                                Methods Enzymol.                                                              183: 63-98; and                                                               ESTs: fast Identity                                                           =                           functions: fasta, tfasta, fastx, tfastx, and ssearch. Smith, T. F. and                                                          M. S. Waterman                                                                (1981) Adv. 95% or                                                            greater and                  Appl. Math. 2:482-489. Match length = 200                                      bases or greater; fastx                                                       E value = 1.0E-8 or                                                           less Full Length                                                              sequences: fastx                                                              score = 100 or greater                                                     BLIMPS A BLocks IMProved Searcher that matches a sequence Henikoff, S                                                            and J. G. Henikoff,                                                           Nucl. Acid Score =                                                            1000 or                     against those in BLOCKS and PRINTS databases to search Res., 19:6565-72                                                         , 1991. J. G.                                                                 Henikoff and S.                                                               greater; Ratio of                                                               for gene families,                                                          sequence homology,                                                            and structural                                                                Henikoff (1996)                                                               Methods Enzymol.                                                              266:88- Score/Strengt                                                         h = 0.75                    fingerprint regions. 105; and Attwood, T. K. et al. (1997) J. Chem. or                                                          larger; and                  Inf. Comput. Sci. 37: 417-424. Probability value =                             1.0E-3 or less                                                             PFAM A Hidden Markov Models-based application useful for Krogh, A. et                                                            al. (1994) J. Mol.                                                            Biol., 235: Score =                                                           10-50 bits,                 protein family search. 1501-1531; Sonnhammer, E. L. L. et al. (1988)                                                            depending on                                                                     Nucleic Acids                                                              Res. 26: 320-322.                                                             individual protein                                                                families                                                                   ProfileScan An                                                               algorithm that                                                                searches for                                                                  structural and                                                                sequence Gribskov,                                                            M. et al. (1988)                                                              CABIOS 4:61-66;                                                               Score = 4.0 or                                                                greater                     motifs in protein sequences that match sequence patterns Gribskov, et                                                           al. (1989) Methods                                                            Enzymol.                    defined in Prosite. 183:146-159; Bairoch, A. et al. (1997) Nucleic                                                                 Acids Res. 25:                                                             217-221.                   Phred A base-calling algorithm that examines automated Ewing, B. et al.                                                          (1998) Genome                                                                   sequencer traces                                                            with high sensitivity                                                          and probability.                                                             Res. 8:175-185;                                                               Ewing, B. and P.                                                                 Green (1998)                                                               Genome Res. 8:186-194                                                         .                          Phrap A Phils Revised Assembly Program including SWAT and Smith, T. F.                                                           and M. S. Waterman                                                            (1981) Adv. Score =                                                           120 or greater;                                                                 CrossMatch,                                                                 programs based on                                                             efficient implementat                                                         ion of Appl. Math.                                                            2:482-489; Smith, T.                                                          F. and M. S. Match                                                            length = 56 or                                                                  the Smith-Waterman                                                          algorithm, useful in                                                          searching Waterman                                                            (1981) J. Mol. Biol.                                                          147:195-197; greater        sequence homology and assembling DNA sequences. and Green, P.,                                                                  University of                                                                 Washington,                  Seattle, WA.                                                                Consed A graphical tool for viewing and editing Phrap assemblies                                                                 Gordon, D. et al.                                                             (1998) Genome                                                                    Res. 8:195-202.                                                             SPScan A weight                                                              matrix analysis                                                               program that scans                                                            protein Nielson, H.                                                           et al. (1997)                                                                 Protein Engineering                                                           Score = 5 or greater        sequences for the presence of secretory signal peptides. 10:1-6;                                                                Claverie, J. M. and                                                           S. Audic (1997)                                                                  CABIOS 12:                                                                 431-439.                   Motifs A program that searches amino acid sequences for patterns                                                                 Bairoch et al.                                                                supra; Wisconsin                                                                that matched those                                                          defined in Prosite.                                                           Package Program                                                               Manual, version                                                                  9, page M51-59,                                                            Genetics Computer                                                                Group, Madison,                                                            WI.                      __________________________________________________________________________

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 3                                        - - <210> SEQ ID NO 1                                                        <211> LENGTH: 249                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE: -                                                              <223> OTHER INFORMATION: 1887516                                               - - <400> SEQUENCE: 1                                                         - - Met Asp Leu Leu Leu Asn Lys Asn Ser Leu Pr - #o Pro Pro Ile Val           1               5 - #                 10 - #                 15              - - Val Thr Tyr His Ser Thr Tyr Leu Ile Ser Gl - #u His Leu His Gly                           20 - #                 25 - #                 30              - - Ser Leu Ile Phe Val Leu Phe Gly Phe Phe Me - #t Tyr Phe Phe Leu                           35 - #                 40 - #                 45              - - Leu Leu Leu Phe Phe Asn Val Gln Arg Leu Al - #a Phe Pro Phe Gly                           50 - #                 55 - #                 60              - - Ile Pro Asn Asp Pro Met Leu Trp Ser Glu Gl - #y Gln Ser His Leu                           65 - #                 70 - #                 75              - - Cys Trp Arg Ser Pro Leu Ile Pro Ser Ala Gl - #n Phe Arg Gly Ser                           80 - #                 85 - #                 90              - - Arg Ala Asp Ile Arg Gly Ser Met Leu His Se - #r Ser Ser Gly Arg                           95 - #                100 - #                105              - - Val Val Pro Leu Asn Pro Ala Thr Lys Leu Se - #r Pro Leu Glu Ser                          110  - #               115  - #               120              - - Gln Met Ala Leu His Thr Arg Ala Val Glu Al - #a Gly Met Val Phe                          125  - #               130  - #               135              - - Gly His Arg Ala Glu His Lys Asp Pro Arg Se - #r Val Trp Glu Ser                          140  - #               145  - #               150              - - Tyr Trp Leu Leu Gly Ser Pro Trp Ala Glu Va - #l Thr Arg Leu His                          155  - #               160  - #               165              - - Pro Arg Arg Ala Gln Leu Gly Ser Leu Pro Pr - #o Pro Asp Pro Arg                          170  - #               175  - #               180              - - Thr Thr His Arg Arg Gly Ala Val Ser Ile Ph - #e Leu Lys Gly Pro                          185  - #               190  - #               195              - - Phe Gly Asp Leu Val Leu Ser Val Glu Arg Th - #r Asp Val Ala Leu                          200  - #               205  - #               210              - - Ser Ser Gln His Ile Pro Gly Ser Gly Arg Pr - #o Gln Leu Lys Gln                          215  - #               220  - #               225              - - Cys Gln Gly Pro Gln Gly Ser His Leu Asp Ar - #g Pro Thr Ala Cys                          230  - #               235  - #               240              - - Asn Ser Ala Leu Leu Arg Arg Gln His                                                      245                                                            - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 1205                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE: -                                                              <223> OTHER INFORMATION: 1887516                                               - - <400> SEQUENCE: 2                                                         - - ctcctccctg ctgcttggga gttcacccag gagagcgaaa tgggtgtgtt tt -             #gttaaggt     60                                                                 - - cccttgtgga gactcagggc tgaatcctgc ttggtaatat cagtgtgtgt gc -            #ttggggat    120                                                                 - - ggaccttcta ctgaataaaa actccctccc tccccccatt gtggtcacat at -            #cattctac    180                                                                 - - atatctcatc tctgagcatc tccatggaag cttgattttt gttctttttg gt -            #ttctttat    240                                                                 - - gtattttttt ctgttgttat tattttttaa tgttcaaaga ctagcctttc cc -            #tttgggat    300                                                                 - - tccaaatgat cccatgctgt ggtctgaggg gcaaagccac ctatgttggc gc -            #tcgccatt    360                                                                 - - aatccccagc gctcagttta gaggctcacg tgcagacatc agaggctcca tg -            #ctgcacag    420                                                                 - - tagctcaggc agggtagtgc ctctcaaccc agccacaaaa ctctccccgc tg -            #gagtccca    480                                                                 - - gatggcgctt cacaccaggg cagtggaggc aggcatggtt tttgggcaca gg -            #gcagagca    540                                                                 - - taaggatccc aggtcagtgt gggagagcta ctggctctta ggatcacctt gg -            #gcagaagt    600                                                                 - - cacacggctt catcctagga gggcccagct tgggagtctg cctccccctg at -            #cccaggac    660                                                                 - - cacccacagg agaggggcag tgtccatctt tctgaaggga ccctttggag at -            #ctcgtcct    720                                                                 - - aagtgtggag aggactgacg tggccctgtc atctcaacac atcccagggt ca -            #ggcaggcc    780                                                                 - - tcagctgaaa caatgtcagg gtcctcaagg gtcccattta gacagaccca cg -            #gcttgtaa    840                                                                 - - cagtgcgctc ctcaggaggc agcactagcg catacccact ccccacggac ac -            #tgagttcc    900                                                                 - - tggtgacagc tgcagcccca gccccgccag gagtcctgga gacagcagcc ct -            #cagagacc    960                                                                 - - ctgcaggagt gagtgcaccc caccttgctc agccacaccc cactcccctg tg -            #ccctgtag   1020                                                                 - - ttgtgctgcc catgctccac acaccatggg gcccctttgc tcatttttgg ac -            #tatttata   1080                                                                 - - cagcaggttt ggatcatgtt tttctactaa taagaatgct aacattgttg tg -            #tagataat   1140                                                                 - - cagtgagggc tttatgaagt ttacaccttt gcattattaa aggaaataac ag -            #ttaaaaaa   1200                                                                 - - aaaaa                 - #                  - #                  -      #          1205                                                                  - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 255                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE: -                                                              <223> OTHER INFORMATION: g1006572                                              - - <400> SEQUENCE: 3                                                         - - Met Asn Phe Ile Ile Asn Ser Pro Leu Glu Gl - #n Phe Thr Thr Arg           1               5 - #                 10 - #                 15              - - Val Tyr Phe Gly Leu Ser Ser Gly Leu Ile As - #n Leu Asp Thr Met                           20 - #                 25 - #                 30              - - Thr Leu Thr Ser Phe Ser Met Tyr Ser Ile Al - #a Val Val Ala Leu                           35 - #                 40 - #                 45              - - Met Leu Gly Phe Ser Ile Leu Asn Asp Asn As - #n Thr Asn Ile Leu                           50 - #                 55 - #                 60              - - Pro Thr Arg Trp Ser Leu Ala Phe Glu Ser Le - #u Tyr Phe Thr Val                           65 - #                 70 - #                 75              - - Glu Lys Met Val Ser Glu Gln Met Gly Gly Le - #u Glu Gly Arg Leu                           80 - #                 85 - #                 90              - - Leu Phe Pro Phe Met Phe Ser Leu Phe Met Ty - #r Ile Leu Met Ala                           95 - #                100 - #                105              - - Asn Val Val Ser Leu Val Pro Tyr Ser Tyr Al - #a Ile Asn Ala Gln                          110  - #               115  - #               120              - - Leu Met Trp Thr Met Gly Leu Ser Val Ala Il - #e Trp Ile Gly Cys                          125  - #               130  - #               135              - - Thr Leu Thr Gly Leu Ala Asn His Gly Ala Ly - #s Phe Phe Gly Leu                          140  - #               145  - #               150              - - Phe Leu Pro Ser Gly Thr Asn Leu Pro Leu Va - #l Pro Val Leu Val                          155  - #               160  - #               165              - - Met Ile Glu Leu Leu Ser Tyr Met Ala Arg Al - #a Leu Ser Leu Gly                          170  - #               175  - #               180              - - Leu Arg Leu Gly Ser Asn Ile Leu Ala Gly Hi - #s Leu Leu Leu Val                          185  - #               190  - #               195              - - Ile Leu Ala Gly Leu Ile Leu Asn Phe Ile Se - #r Ile Ser Met Phe                          200  - #               205  - #               210              - - Thr Phe Ala Leu Gly Ile Leu Pro Leu Ser Il - #e Leu Leu Gly Ile                          215  - #               220  - #               225              - - Val Ala Leu Glu Ser Ala Ile Ala Phe Ile Gl - #n Ala Met Val Phe                          230  - #               235  - #               240              - - Thr Met Leu Thr Cys Ser Tyr Ile Lys Asp Al - #a Ile His Leu His                          245  - #               250  - #               255            __________________________________________________________________________

What is claimed is:
 1. A substantially purified polypeptide comprisingthe amino acid sequence of SEQ ID NO:1.
 2. A pharmaceutical compositioncomprising the polypeptide of claim 1 in conjunction with a suitablepharmaceutical carrier.